ATPase assay

F₁ samples were diluted 1:100 in buffer A to a concentration of ∼7 nM and incubated with 0.1 mM ATP for 20 min at room temperature (For the photoswitching experiment shown in Fig. 3 the protein was diluted 1:50). The increase in concentration of inorganic phosphate (P_i) in the sample was assayed using a Malachite Green Phosphate Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA). The absorption at 620 nm, indicative of the presence of the green molybdophodphoric acid complex was measured against buffer with a UV-VIS spectrometer (IMPLEN Nanophotometer, München, Germany). The P_i concentration of the sample was calculated from the 620 nm absorption of the sample using a calibration curve measured with buffer A supplemented with P_i at known concentrations. Under the tested conditions the ATP hydrolysis activity of the double cysteine F₁ mutants was at least 50% of the parent cysteine-less F₁ construct.

(A) SDS-PAGE gels of the mutant before and after incubation with ABDM. The appearance of a shifted high molecular weight band indicates the formation of crosslinks between the α and β subunit (crosslinking ratio: 0.3). (B) F₁ catalyzed increase of the P_i concentration after ATP incubation for a trans-ABDM crosslinked F₁ mutant sample after subsequent illumination with UV and blue light. The data refer to three experiments involving independent crosslinking, illumination and ATPase activity assay. The sample shown in the right lane of the SDS-PAGE gel in panel A refers to the red trace (spheres).